Action potentials are typically initiated by sodium channels (Navs). This role is shared by Navs in many tissues, and mutations or other dysfunction of Navs lead to tissue specific diseases, including arrythmias, epilepsy, and chronic neuropathic pain. A key similarity in all channelopathies is that an insult to the basic architecture of the channel leads to a change in function. Determining this basic architecture at atomic resolution and the stereo-chemical details of selectivity, gating, and of anti-hypertension drug binding are the purposes of this proposal. While structural studies of K+ channels are quite advanced, there are currently no analogous studies and no stereo-chemical understanding of Na+ and Ca2+ selectivity or of drug binding. The broad aim of this proposal is to determine the mechanisms of sodium and calcium selectivity and anti-hypertension drug binding in a family of voltage-gated ion channels. The first specific aim will complete our current structure, the second specific aim will determine the mechanism of ion selectivity for both Na+ and Ca2+, and the third aim will develop and utilize methods to stabilize inherently flexible molecules. Multiple antibodies and engineered disulfide bonds will be used to stabilize specific gating states. These states will be confirmed by structural studies, and their structures will be determined by x-ray crystallography. The long-term benefit of this work will be a unifying model for the hundreds of disease causing mutations, a stereo-chemical view of drug binding leading to improved therapeutics, a better understanding of ion selectivity and the relationship between Navs and Cavs, and a physical model for channel gating. An additional benefit will be the development of methods for immobilizing inherently flexible molecules to aid in crystallization. This project will use x-ray crystallography to determine the first Na+ channel structure. The public health benefit will be improved treatment methods for Na+ channel dysfunction resulting from improved anti- arrhythmias drugs.